Aziridinyl quinone antitumor agents

ABSTRACT

The use of aziridinyl quinones as antitumor agents is disclosed. A compound which has been found to be particularly effective is the compound 2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone. Treatment is described in connection with various forms of leukemia, for example, as well as B16 melanoma, Lewis lung carcinoma, and the ependymoblastoma brain tumor system.

This is a continuation of application Ser. No. 098,845, filed Nov. 30,1979 which in turn is a continuation of application Ser. No. 954,218,filed Oct. 24, 1978, which in turn is a divisional of application Ser.No. 758,812, filed Jan. 12, 1977. Application Ser. Nos. 758,812, 954,218and 098,845 are all now abandoned.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to the use of aziridinyl quinones aspotential central nervous system antitumor agents. More particularly,the present invention is concerned with adiaziridinyldi(carboethoxyamino)benzoquinone which has been found tohave significant activity as a central nervous system antitumor agent.

A recent analysis of murine antitumor test data has indicated that theaziridinylquinones, as a family, possess significant activity againstlymphoid leukemia L1210 as well as other test systems. While the L1210results were all obtained on an intraperitoneally (IP) implanted tumor,the molecular properties of the compounds appeared to fit some of therequirements suggested by Rall and Zubrod in Annu. Rev. Pharmacol., 2,109 (1962) as important for central nervous system (CNS) penetration.Subsequent testing of these compounds in several intracerebral (IC)tumor systems indicated that the aziridinylquinones possessedsubstantial IC antitumor activity.

However, a major problem associated with almost all of the antitumoractive aziridinylquinones is the very low aqueous solubility of thecompounds. This has greatly complicated the preparation of a suitableparenteral dosage form. The present invention is concerned withaziridinylquinones having optimized properties as CNS antitumor agents.Emphasis has been placed on the study of the effect of nonionicfunctional groups, since ionic materials have difficulty penetrating theblood-brain barrier. The chemistry used in the synthesis of thecompounds of interest is based mainly on the reactions oftetrachlorobenzoquinone, also known as chloranil, and itstetrafluoroanalog, fluoranil. Chloranil may be obtained in a well knownmanner from phenol, p-chlorophenol or p-phenylenediamine by treatmentwith potassium chlorate and hydrochloric acid. Fluoranil is preparedfrom chloranil by reaction with calcium fluoride at elevatedtemperatures.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The reaction of chloranil with aziridine and its analogs was carried outto produce the disubstituted compounds Nos. 2 and 3, as shown in Table Iby a procedure as set forth in British Pat. No. 762,723. Compound 4,2,5-dichloro-3,6-diaziridinyl-1,4-benzoquinone, was converted to thecorresponding ethyl- (No. 5) and propyl mercapto (No. 6) derivatives. Itwas possible to replace all the chlorine atoms in chloranil withethylmercapto groups, No. 7. Chloranil also reacted with the sodium saltof urethane to produce No. 8, a key intermediate. Attempts to reactchloranil with methyl carbamate, urea, thiourea, and diethyl malonate ina similar manner were not successful. The diaziridinylbenzoquinoneurethanes, Nos. 9-11, were prepared from No. 8. The aziridine rings ofNo. 9 were opened with HCl to give the bis(2-chloroethylamine)derivative, No. 12. The preparation of compound No. 9 is as follows:

                                      TABLE I                                     __________________________________________________________________________    PHYSICAL AND CHEMICAL DATA                                                     ##STR1##                                                                                             Yield,                                                                            λ.sub.max.sup.CH.sbsp.3.sup.OH             No.                                                                              R.sub.1 R.sub.2 Mp, °C.                                                                     %   (log ε)                                                                      Mol formula                                                                           Analyses.sup.f                     __________________________________________________________________________     2 MeAz.sup.a                                                                            Cl      .sup. 155.sup.b                                                                    52  347 (4.24)                                                                           C.sub.12 H.sub.12 Cl.sub.2 N.sub.2                                            O.sub.2 C, H, N, Cl                         3 Me.sub.2 Az                                                                           Cl      200  15  362 (4.30)                                                                           C.sub.14 H.sub.16 Cl.sub.2 N.sub.2                                            O.sub.2 C, H, N, Cl                         5 Az      SC.sub.2 H.sub.5                                                                      .sup. 135.sup.c                                                                    33  340 (6.18)                                                                           C.sub.14 H.sub.18 N.sub.2 O.sub.2                                             S.sub.2 C, H, N, S                          6 Az      SC.sub.3 H.sub.7                                                                      159  44         C.sub.16 H.sub.22 N.sub.2 O.sub.2                                             S.sub.2 C, H, N, S                          7 SC.sub.2 H.sub.5                                                                      SC.sub.2 H.sub.5                                                                       88  13         C.sub.14 H.sub.20 O.sub.2 S.sub.2                                                     C, H, S                             8 NHCOOC.sub.2 H.sub.5                                                                  Cl      220  27  317 (4.06)                                                                           C.sub.12 H.sub.12 Cl.sub.2 N.sub.2                                            O.sub.6 C, H, N, Cl                         9 Az      NHCOOC.sub.2 H.sub.5                                                                  .sup. 230.sup.d                                                                    68  340 (4.17)                                                                           C.sub.16 H.sub.20 N.sub.4 O.sub.6                                                     C, H, N                            10 MeAz    NHCOOC.sub.2 H.sub.5                                                                  198  89         C.sub.18 H.sub.24 N.sub.4 O.sub.6                                                     C, H, N                            11 Me.sub.2 Az                                                                           NHCOOC.sub.2 H.sub.5                                                                  175  22  355 (4.23)                                                                           C.sub.20 H.sub.30 N.sub.4 O.sub.6                                                     C, H, N                            12 NHCH.sub.2 CH.sub.2 Cl                                                                NHCOOC.sub.2 H.sub.5                                                                  215  68  340 (4.40)                                                                           C.sub.16 H.sub.22 Cl.sub.2 N.sub.4                                            O.sub.6 C, H, N, Cl                        15 Az      Br      .sup. 181.sup.e                                                                    47  347 (4.15)                                                                           C.sub.10 H.sub.8 Br.sub.2 N.sub.2                                             O.sub.2 C, H, N                            16 MeAz    Br      160  69  357 (4.24)                                                                           C.sub.12 H.sub.12 Br.sub.2 N.sub.2                                            O.sub.2 C, H, N                            17 Me.sub.2 Az                                                                           Br      210  16  362 (4.26)                                                                           C.sub.14 H.sub.18 Br.sub.2 N.sub.2                                            O.sub.2 C, H, N                            18 NHNH.sub.2                                                                            Cl      198  78  330 (3.59)                                                                           C.sub.6 H.sub.8 Cl.sub.4 N.sub.4                                              O.sub.2 C, H, N, Cl                        19 NH.sub.2                                                                              Cl      360  72         C.sub.6 H.sub.4 Cl.sub.2 N.sub.2                                              O.sub.2 C, H, N, Cl                        21 Me.sub.2 Az                                                                           NHCOCH.sub.3                                                                          205  73         C.sub.18 H.sub.26 N.sub.4 O.sub.4                                                     C, H, N                            22 NCHOC.sub.2 H.sub.5                                                                   Cl      148  76  320 (5.40)                                                                           C.sub.12 H.sub.12 Cl.sub.2 N.sub.2                                            O.sub.4 C, H, N, Cl                        __________________________________________________________________________     ##STR2##                                                                      .sup.b Lit. mp 182°.                                                   .sup.c Lit. mp 134°.                                                   .sup.d Lit. mp 250°.                                                   .sup.e Lit. mp 178°.                                                   .sup.f Analyses indicated are determined experimentally to be within          ±0.4% of theoretically calculated values.                             

2,5-DIAZIRIDINYL-3,6-BIS(CARBOETHOXYAMINO)-1,4-BENZOQUINONE, NO. 9General Procedure for Nos. 10, 11, and 21

A solution of No. 8 (1.5 g, 0.004 mol) in tetrahydrofuran (THF) (60 ml)was added dropwise to a stirred solution of ethylenimine (1.0 ml, 0.03mol) and triethylamine (2.5 ml) in THF (20 ml). After the addition wascomplete (0.5 hr), the reaction mixture was stirred at room temperatureovernight, about 16 hrs. Excess solvent was removed in vacuo and theresulting reddish-brown solid was washed with ice-cold water and driedover KOH pellets in vacuo. Recrystallization from ethanol gave 1.06 g(68%) of orange needles: mp 230° dec (lit. mp 250°). Compound No. 9 isknown in the patent literature, being disclosed in U.S. Pat. No.2,913,453.

The dibromodiaziridinylbenzoquinones, Nos. 15-17 as shown in Table I,were prepared in a three-step synthesis starting from benzoquinone.Reaction of 1,4-benzoquinone with anhydrous zinc chloride in methanolgave No. 13, 2,5-dimethoxy-1,4-benzoquinone, which was brominated toyield No. 14, 2,5-dibromo-3,6-dimethoxy-1,4-benzoquinone. Aziridinereplaced the methoxy groups rather than the halogen atoms in accordancewith the reported relative reactivities of tetrasubstitutedbenzoquinones.

Ammonia and hydrazine reacted with chloranil to produce Nos. 18 and 19.These compounds were unreactive toward other nucleophiles because ofring deactivation by the electron-donating amino groups. Reactivation ofthe ring by acetylation produced No. 20,2,5-dichloro-3,5-acetylamino-1,4-benzoquinone, which then reacted with2,2-dimethylaziridine to give No. 21. The monomethyl and unsubstitutedaziridine analogs of No. 21 had previously been prepared and tested.Compound No. 19 also reacted with diethoxymethyl acetate to give No. 22but this compound was unreactive toward aziridine. Physical and chemicaldata for these compounds are summarized in Table I.

So far as antitumor activity is concerned, chloranil itself, as well ascompounds Nos. 4, 14, 19, and 20 had had prior testing and were inactivein the L1210 system. Compounds Nos. 2, 3, 5-12, 15-18, 21, and 22 weretested during this study in the lymphoid leukemia L1210 system bystandard NCI protocols on the Q4D (day 1, 5, 9 with a total of 3injections) and the QD1-9 (day 1-9 with a total of 9 injections)treatment schedules. Among these compounds, only compound No. 9 wasactive with an increase in life span (ILS) >25%, as shown in Table II.

The optimum dose and activity (%ILS) for No. 9 on the Q4D schedule was6.25 mg/kg (54%). Compound No. 9 had greater L1210 activity on the QD1-9schedule, however (Table II). The compound had an aqueous solubility ofapproximately 0.5 mg/ml. Using the assumptions and constants ofFreireich et al., Cancer Chemother. Rep., 50, 219 (1966), and an optimumdose of 2 mg/kg in the mouse, an average human maximum tolerated dose of10 mg per dose can be approximated. An active dose range of 2.5-10 mgper dose can be estimated. Since only 20 ml of water would be requiredto dissolve 10 mg. of No. 9, the solubility of this compound appears tobe in the acceptable range.

                                      TABLE II                                    __________________________________________________________________________    ANTITUMOR ACTIVITY.sup.a OF COMPOUND 9                                        210 lymphoid leukemia.sup.b                                                                P388 lymphocytic leukemia.sup.b                                                             B16 melanocarcinoma.sup.b                                                                 Lewis lung carcinoma.sup.c             Expt         Expt          Expt        Expt                                   no.                                                                              Dose.sup.d                                                                        ILS.sup.e                                                                        T-C.sup.f                                                                        no. Dose                                                                             ILS:                                                                              T-C                                                                              no.                                                                              Dose                                                                             ILS                                                                              T-C                                                                              no.                                                                              Dose                                                                             ILS:                                                                             T-C                           __________________________________________________________________________    42 12.50                                                                             T.sup.g                                                                          -5.0                                                                             4323                                                                              3.12                                                                              12 -3.6                                                                             267                                                                              625                                                                              T  -3.5                                                                             8  4.00                                                                             T  -0.1                             6.25                                                                              27 -4.1   1.56                                                                             138 -2.4  3.12                                                                             58 -1.9  2.00                                                                             38 -0.3                             3.12                                                                              113                                                                              -3.2   0.78                                                                             111 -2.4  1.56                                                                             38 -1.2  1.00                                                                             27 -0.1                                                        0.78                                                                             36 -1.4                                      40 4.60                                                                               0 -4.6                                                                             4328                                                                              6.25                                                                             T   -6.1                                                                             284                                                                              6.25                                                                             T  -4.4                                                                             9  4.00                                                                             T  + 1.7                            3.12                                                                              28 -3.1   3.12                                                                              27 -4.1  3.12                                                                             43 -2.7  2.00                                                                             34 +1.6                             2.00                                                                              101                                                                              -3.6   1.56                                                                             135 -4.0  1.56                                                                             44 -3.6  1.00                                                                              0 +2.4                                           0.78                                                                              99 -2.1  0.78                                                                             13 -2.2                                      62 1.30                                                                              60 -2.3                                                                             4387                                                                              3.12                                                                              0  -3.4                                                                             285                                                                              6.25                                                                             T  -3.5                                         0.88                                                                              32 -1.4   1.56                                                                             112 -2.1  3.12                                                                             70 -1.2                                                       0.78                                                                             112 -1.4  1.56                                                                             58 -2.7                                                                    0.78                                                                             36 -1.1                                      95 4.60                                                                              51 -3.9                                                                   3.12                                                                              169                                                                              -4.1                                                                   2.00                                                                              125                                                                              -3.8                                                                __________________________________________________________________________     Protocols and tumor systems described in Geran et al., Cancer Chemother.      Rep., Part 3, 3(No. 2), 1 (1972).                                             .sup.b Ip tumor implantation, ip QD19 treatment schedule.                     .sup.c Subcutaneous tumor implantation, ip QD515 treatment schedule.          .sup.d mg/kg/injection.                                                       .sup.e ILS = [(treated survival + control survival) × 100%]  - 100%      .sup.f T-C = average weight change of test group minus average weight        change of control animals in grams on day 5.                                  .sup.g T = toxic dose.                                                   

                                      TABLE III                                   __________________________________________________________________________    INTRACEREBRAL ANTITUMOR ACTIVITY.sup.a OF COMPOUND 9                          Ic L1210     Ic P388                                                          lymphoid leukemia.sup.b                                                                    lymphocytic leukemia.sup.b                                                                Ic ependymoblastoma.sup.c                            Expt         Expt        Expt                                                 no.                                                                              Dose.sup.d                                                                        ILS.sup.e                                                                        T-C.sup.f                                                                        no.                                                                              Dose                                                                             ILS                                                                              T-C                                                                              no.                                                                              Dose                                                                             ILS                                                                              T-C                                                                              Cures.sup.g                              __________________________________________________________________________    28 6.25                                                                              10 -2.1                                                                             25 6.25                                                                             T.sup.h                                                                          -2.1                                                                             133                                                                              4.00                                                                             T  -3.0                                           3.12                                                                              84 -1.1  3.12                                                                             39 -0.1  2.00                                                                             349                                                                              -1.3                                                                             4/6                                         1.56                                                                              51 -0.6  1.56                                                                             37 -0.6  1.00                                                                             348                                                                              -1.3                                                                             3/6                                         0.78                                                                              16 +0.5  0.78                                                                             27 +1.0                                                                    0.39                                                                              7 +0.8                                                    29 6.25                                                                               3 -2.8                                                                             26 6.25                                                                             T.sup.                                                                           -2.8                                                                             136                                                                              8.00                                                                             T  -5.0                                           3.12                                                                              79 -2.1  3.12                                                                             31 -2.1  4.00                                                                             276                                                                              -2.4                                                                             4/6                                         1.56                                                                              46 -0.5  1.56                                                                             51 -1.1  2.00                                                                             277                                                                              -1.1                                                                             5/6                                         0.78                                                                               3 -0.5  0.78                                                                             25 -0.4  1.00                                                                             126                                                                              -0.2                                                                             1/6                                                      0.39                                                                              2 -0.7                                                                             144                                                                              4.00                                                                             T  -3.9                                                                    2.00                                                                              71                                                                              -2.7                                                                             5/6                                                                  1.00                                                                             171                                                                              -2.1                                                                             5/6                                                                  0.50                                                                              62                                                                              -1.8                                                                    0.25                                                                              30                                                                              -1.5                                        __________________________________________________________________________     .sup.a Protocols and tumor systems described in Geran et al., Cancer          Chemother. Rep., Part 3, 3(No. 2), 1 (1972).                                  .sup.b Ic tumor implantation, ip QD19 treatment schedule.                     .sup.c Ic tumor implantation, ip QD15 treatment schedule.                     .sup.d mg/kg/injection.                                                       .sup.e See Table II.                                                          .sup.f T-C C = average weight change of test group minus average weight       change of control animals in grams on day 5.                                  .sup.g Number of animals alive per six test animals on day of termination     of experiment (day 99 for expt 133 and day 60 for expt 136 and 144).          .sup.h Toxic dose.                                                       

                                      TABLE IV                                    __________________________________________________________________________    ANTITUMOR ACTIVITY OF COMPOUND 9.sup.1                                        (IC)Ependymo-                                                                 blastoma.sup.2                                                                         (IP)L1210                                                                            (IC)L1210                                                                            (IP)B16                                                                              (IP)P388                                                                             (IC)P388                                 OD.sup.3                                                                         % ILS OD % ILS                                                                             OD % ILS                                                                             OD % ILS                                                                             OD % ILS                                                                             OD % ILS                                 __________________________________________________________________________    2   >277(5)*                                                                           3  169 3  84  3  94  1.6                                                                              138(1)                                                                            1.5                                                                              51                                    1  >202(4)                                                                             3  113 3  79  3  70  1.6                                                                              135 3  39                                    2  >172(6)                                                                             2  101 5  63  3  62  0.8                                                                              112(1)                                       1  >171(5)                                                                             4  100 3  58                                                         __________________________________________________________________________     Notes:                                                                        .sup.1 Treatment schedules are QD19 unless otherwise indicated                .sup.2 QD1-5 treatment schedule                                               .sup.3 mg/kg/injection                                                        *Numbers in parentheses are survivors of 6 test mice on experiment            termination day (EMday 60; P388day 30).                                  

Because of its activity in the L1210 system, No. 9 was tested in someadditional intraperitoneal (IP) and intracerebral (IC) tumor models.Biological data are shown for the L1210 (QD1-9 schedule) and P388leukemia systems plus the B16 melanocarcinoma and Lewis lung solid tumorsystems in Table II. Central nervous system antitumor activity is givenin Table III for the intracerebrally implanted L1210, P388, andependymoblastoma tumor systems. The criterion for minimum activity isdefined here as follows: intraperitoneal and intracerebral L1210leukemia, 25%; intraperitoneal and intracerebral P388 leukemia, 25%; B16melanoma, 40%; Lewis lung carcinoma, 25%; and intracerebralependymoblastoma, 40%. A summary of significant antitumor data obtainedwith compound No. 9 is shown in Table IV.

The L1210, P388 and B16 test systems described in Table II utilizeintraperitoneal (IP) tumor implantation and IP drug treatment while theLewis lung system employs subcutaneous tumor implantation and IPtreatment. All three CNS tumor models (Table III) use intracerebral (IC)tumor implantation and IP treatment.

Table II indicates that thediaziridinyldi(carboethoxyamino)benzoquinone, No. 9, has reproducibleactivity in excess of ILS 100% in the standard IP L1210 test on theQD1-9 treatment schedule. The optimum dose (OD) is 2-3 mg/kg and atherapeutic ratio (TR, highest active dose divided by lowest activedose) of approximately 4 is seen. Similar activity results were observedin the P388 system at a slightly lower OD (1-2 mg/kg). B16 melanomaactivity was significant with activity as high as 70% observed at an ODof 3.12 mg/kg. Marginal activity was seen in the refractory Lewis lungtumor system. While insufficient low-dose testing was carried out toestablish TI values in the last three systems, No. 9 might becharacterized as a reasonably toxic material with a TR in the range of3-4.

Antitumor activity was observed with compound No. 9 in all of the ICtumor systems studied (Table III). While only two dose responseexperiments were conducted in the IC L1210 and IC P388 systems,reproducible activity was seen in both cases. The activity of No. 9 inIC L1210 (ILS 79, 84% at OD 3.12 mg/kg) is noteworthy.

A third IC tumor model, the ependymoblastoma system, was also studied.This system requires intracerebral implantation of a solid tumorfragment while the IC L1210 and IC P388 systems utilize IC inoculatedascites tumor fluid. All three use IP drug treatment however. CompoundNo. 9 was very active in the ependymoblastoma IC tumor model. Cures(survivors from the group of six mice on the last day of the experiment)are indicated. Experiments 136 and 144 were terminated on day 60. Sincethe number of cures was about the same, the differences in T/C values inthese two experiments reflect differences in the life span of the twodifferent sets of control animals. Experiment 133 was terminated on day99 with a resulting higher activity value. A majority of the testanimals were alive on day 99 at a dose of 2.0 mg/kg in this experiment.An optimum dose of 1-2 mg/kg is indicated for No. 9 in theependymoblastoma system with a TR of 4-8.

The inactivity of the other compounds studied here is also noteworthy.Methyl substitution in the aziridine ring (Nos. 10, 11, 21) abolishedactivity relative to No. 9 and a previously reportedaziridinylacetylaminobenzoquinone. All ring-halogenatedaziridinylquinones (Nos. 2-4, 15-17) were inactive. Although thediethylmercapto derivative No. 5 gave one marginally active test (ILS30%, IP L1210, Q4D), the activity was not reproducible. Thedipropylmercapto analog No. 6 was inactive. These results are incontrast with the activities found (ILS 50%) for the correspondingalkoxy aziridinylbenzoquinone compounds. When the aziridine rings of No.9 were opened to the corresponding di (one arm mustard) derivative No.12, activity was abolished.

In the compounds and test systems investigated here, the aziridine ringis necessary, but not sufficient, for antitumor activity. It has beentheorized that the inactivity of the dihalodiaziridinyl compounds is dueto either an electronic effect and resulting effect on the redoxpotential, a lipophilic transport effect, or a steric effect on thedegree of coplanarity of the quinone and aziridine ring systems.

As previously discussed, a first approach utilized chloranil as astarting material and resulted in an aziridinyl quinone urethanederivative, compound No. 9, which was very active in the intraperitonealleukemia L1210, leukemia P388, and the B16 melanoma test systems. Inaddition, substantial intracerebral (IC) L1210 and P388 activity wasobserved and the compound was curative in the murine ependymoblastomabrain tumor model. The solubility of this urethane derivative appears tobe adequate,

Further investigations were initiated to produce the previouslyunevaluated diaziridinyl benzoquinone derivatives containing aminosubstituents in the other two available ring positions.

While 2,5-diaziridinyl-1,4-benzoquinone derivatives may be prepared in anumber of ways, substitution reactions utilizing halo or alkoxy quinoneshave been most generally employed. Chloranil has been used extensivelyas a starting material for the preparation of2,5-diaziridinyl-3,6-disubstituted-1,4-benzoquinones. Although thechlorine atoms in the 2- and 5-positions are readily replaced bynucleophiles, the electron donating properties of the added groupsusually deactivate the ring toward replacement of the other two chlorineatoms. While compound 4, for example, as shown in Table V, is easilyprepared from chloranil and ethylenimine, this compound and otherdiaminodichloro-p-benzoquinone derivatives undergo no furtherdisplacement reactions with amines. This is not the case, however, withthe analogous difluoro analog, compound No. 32, which is prepared fromtetrafluoro-1,4-benzoquinone, also known as fluoranil.

Reactions of this type are described, for example, by Finley, Chemistryof Quinonoid Compounds, Part 2, S. Patai, Ed., Wiley, N.Y., 1974, p.1101; Wallenfals et al., Angew. Chem., 70, 313 (1958); Wallenfals et al.Ann., 667, 55 (1963); and Makarova et al., Zhur. Obshch. Khim., 33, 1643(1963). The two fluorine atoms of compound No. 32 are readily replacedby amines to form tetraamino-benzoquinone derivatives. This reaction wasused as the synthetic method for the preparation of compounds Nos.33-47, shown in Table VI. These agents were evaluated for theirantitumor activity in a number of murine intraperitoneal andintracerebral tumor systems.

                  TABLE V                                                         ______________________________________                                         ##STR3##                                                                                     ##STR4##                                                      ______________________________________                                        23, R.sub.1 = R.sub.2 = Cl                                                                    9, Y = Z = NHCOOC.sub.2 H.sub.5                               24, R.sub.1 = R.sub.2 = F                                                                    27, Y = Z = NHCOCH.sub.3                                        ##STR5##                                                                                     ##STR6##                                                       ##STR7##                                                                                     ##STR8##                                                                      4, Y = Z = Cl                                                                32, Y = Z = F                                                  ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                         ##STR9##                                                                     ______________________________________                                        33, R = NH.sub.2                                                                              41, R = N(CH.sub.3).sub.2                                     34, R = NHCH.sub.3 35, R = NHC.sub.2 H.sub.5 36, R = NHC.sub.3 H.sub.7        37, R = NHC.sub.4 H.sub.9 38, R = NHCH.sub.2 CH.sub.2 OH 39, R                = NHCH.sub.2 CH(OH)CH.sub.2 OH 40, R = NHCH.sub.2 CONH.sub.2                                   ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                    ______________________________________                                    

Although several tetraamino-1,4-benzoquinone derivatives are known, onlythe tetraaziridinyl derivative appears to have been evaluated as anantitumor agent, and its activity in the L1210 leukemia system was onlymoderate (ILS 39%). The selection of the appropriate amino substituentsfor compounds 33-47 was important since they might influence both thebiological transport properties and the chemical reactivity of thecompounds. The partition coefficient, an important factor in determiningthe biological transport properties of a compound, was especiallysignificant since this parameter also gives a rough guide to aqueoussolubility properties which are important in this family of sparinglysoluble compounds. The electronic properties of the R-group substituents(Table VI) should influence the redox potential of the quinone system.This factor is known or postulated to be an important factor indetermining the antitumor activity in other quinone systems, includingmitomycin C analogs and bioreductive alkylating agents. If the redoxpotential should be an important factor in the biological activity ofthe diaziridinylbenzoquinones, the substitution of the other twoavailable positions with alkylamino groups should produce a maximumeffect in the parameter relative to other possible substituents. Thiswould result in either a maximum or minimum in biological activityprovided the activity is a linear function of the redox potential. Thesteric effects of the R-groups should influence the degree ofco-planarity of the ethylenimine and quinone rings. This should affectthe reactivity of the alkylating groups as well as the redox potentialof the quinone. For these reasons, the compounds which were synthesizedfor antitumor evaluation were chosen with the intention of creating aspread in lipophilic (π) steric and electronic R-group effects.

Fluoranil reacted with ethylenimine to produce the key intermediate2,5-diaziridinyl-3,6-difluoro-1,4-benzoquinone. Three isomers arepossible from the disubstitution reaction and two have been previouslyisolated and identified. X-ray crystallographic analysis verified thatcompound No. 32 was the structure of the material used in our reactions.The colorless Konig's adducts sometimes found withβ-hydroxyethylaminobenzoquinones were not observed with the tetraaminoderivatives described here. The physical and chemical properties ofthese compounds are given in Table VII.

Two aziridinylbenzoquinones are presently undergoing laboratory andclinical studies. Trenimon, No. 29, is being studied mainly in Europewhile carbazilquinone, No. 30, has had extensive investigation in Japan.Clinical activity has been reported with both compounds. Compounds Nos.27 and 28 are representative of the amide and alkoxy analogs withintraperitoneal (IP) leukemia L1210 activity.

The urethane derivative, No. 9, has been found to have intracerebral(IC) as well as IP antitumor activity. In an attempt to determine thestructural factors affecting the activity of the diaminodiaziridinylanalogs, the compounds Nos. 33-47 were prepared and evaluated in the IPmurine L1210 lymphoid leukemia tumor model. When an IP L1210 test gaveincrease in life span (ILS) values greater than 50%, the compound wasalso tested in the IC L1210 model. Standard NCI protocols were used, asdescribed, for example, in Cancer Chemother. Repts., Part 3, No. 2, 3,1, (1972).

                                      TABLE VII                                   __________________________________________________________________________    PHYSICAL AND CHEMICAL PROPERTIES OF 2,5-DIAZIRIDINYL-3,6-DIAMINO-1,4-BENZO    QUINONES                                                                            Recrystal-               Approx.                                        Compound                                                                            lization                                                                            Yield                                                                             mp      λCH.sub.3 OH.sub.(log ε)                                              H.sub.2 O Sol.                                 Number                                                                              Solvent.sup.(a)                                                                     (%) (°C.)                                                                      Color                                                                             max    (mg/ml)                                                                             log P.sup.(b)                                                                     Mol formula                                                                          Analyses.sup.(f)              __________________________________________________________________________    26    A     84  179 green                                                                             369 (4.13)                                                                           <0.1      C.sub.14 H.sub.20 N.sub.4                                                     O.sub.2                                                                              C,H,N                         31    B     51  157 gray                                                                              325 (3.88)                                                                           2         C.sub.14 H.sub.16 FN.sub.3                                                    O.sub.3                                                                              C,H,N,F                       33    C     73  220 green      <<0.1     C.sub.10 H.sub.12 N.sub.4                                                     O.sub.2                                                                              C,H,N                         34    A     70  220 green                                                                             374 (4.16)                                                                           <0.1      C.sub.12 H.sub.16 N.sub.4                                                     O.sub.2                                                                              C,H,N                         35    A     57  157 green                                                                             374 (4.20)                                                                           0.5       C.sub.14 H.sub.20 N.sub.4                                                     O.sub.2                                                                              C,H,N                         36    A     60  140 green                                                                             375 (4.17)                                                                           <0.1      C.sub.16 H.sub.24 N.sub.4                                                     O.sub.2                                                                              C,H,N                         37    C     91   95 green                                                                             377 (4.12)                                                                           <0.1      C.sub.18 H.sub. 28 N.sub.4                                                    O.sub.2                                                                              C,H,N                         38    A     48  188 green                                                                             375 (4.08)                                                                           2     -1.48                                                                             C.sub.14 H.sub.20 N.sub.4                                                     O.sub.4                                                                              C,H,N                         39    D     77  273 green                                                                             365 (4.15)                                                                           30    -1.97                                                                             C.sub.16 H.sub.24 N.sub.4                                                     O.sub.6                                                                              C,H,N                         40    D     81  200 green                                                                             365 (3.72)                                                                           0.5       C.sub.14 H.sub.18 N.sub.6                                                     O.sub.4                                                                              C,H,N                         41    E     59  112 green                                                                             455 (3.72)                                                                           10        C.sub.14 H.sub.20 N.sub.4                                                     O.sub.2                                                                              C,H,N                                                 298 (3.77)                                            42    F     74  125 green                                                                             455 (3.67)                                                                           60    -0.38                                                                             C.sub.16 H.sub.24 N.sub.4                                                     O.sub.6                                                                              C,H,N                                                 310 (3.71)                                            43    G     64  160 brown                                                                             440 (3.80)                                                                           0.5       C.sub.18 H.sub.24 N.sub.4                                                     O.sub.6                                                                              C,H,N.sup.(c)                                         274 (3.65)                                            44    G     62  .sup. 180.sup.(d)                                                                 brown                                                                             466 (3.80)                                                                           <0.1      C.sub.20 H.sub.28 N.sub.4                                                     O.sub.2                                                                              C,H,N                                                 299 (3.80)                                            45    G     54  235 red 454 (3.69)                                                                           1         C.sub.20 H.sub.28 N.sub.4                                                     O.sub.4                                                                              C,H,N.sup.(e)                                         300 (3.72)                                            46    B     28  224 brown                                                                             453 (3.20)                                                                           <0.1   0.52                                                                             C.sub.18 H.sub.24 N.sub.4                                                     O.sub.4                                                                              C,H,N                                                 307 (3.42)                                            47    F     64  170 green                                                                             452 (3.71)                                                                           20    -0.75                                                                             C.sub.22 H.sub.34 N.sub.6                                                     O.sub.4                                                                              C,H,N                                                 297 (3.76)                                            __________________________________________________________________________     .sup.(a) A = ethanol, B = benzene, C = water wash, D = methanol wash, E =     hexane, F = toluene, G = THF + hexane                                         .sup.(b) Octanol/H.sub.2 O values determined by C. Hansch and M. Yamakawa     Pomona College.                                                               .sup.(c) N calc., 17.06; found, 16.57                                         .sup.(d) lit. mp 177°-                                                 .sup.(e) N calc, 14.42; found, 13.95                                          .sup.(f) Found microanalytical values for the elements listed corresponde     to calculated values within ± 0.4%.                                   

Aqueous saline solution (0.9%) was used as the drug vehicle. Normally,five doses were employed per experiment with the top dose chosen toproduce toxicity. The lower doses were 50% of each preceeding dose.

Since this family of aminoaziridinyl-1,4-benzoquinones subsequentlyproved to have very good L1210 activity, the members were subjected totesting in two additional intraperitoneal systems, including P388lymphocytic leukemia and B16 melanocarcinoma, as well as the murineependymoblastoma brain tumor system. Intraperitoneal antitumor data aregiven in Tables VIII and IX. Table X lists the antitumor activity ofthis series against the intracerebrally implanted solid ependymoblastomaand ascites leukemia L1210 tumor systems. The criterion for minimumactivity, %ILS, is defined here as follows: intraperitoneal andintracerebral leukemia L1210 and intraperitoneal leukemia P388, 25%;intraperitoneal B16 melanoma and intracerebral ependymoblastoma, 40%.Consistent with NCI antitumor test protocols, intraperitoneal (IP)administration of the drug was employed with all five tumor systemsinvestigated. Tumor implantation was IP in the systems designated IPleukemia L1210 and P388 as well as B16 melanoma. Intracerebral (IC)tumor implantation was utilized in the IC L1210 and ependymoblastomatumor systems. Table XI lists those compounds with outstanding activityin two or more tumor systems.

The initial antitumor experiments were carried out in the IP L1210system on the Q4D (day 1, 5, 9) treatment schedule. These experimentsconfirmed the L1210 activity in this series and established the properdose ranges for the determination of any schedule dependency in the IPL1210 system (Table VIII). The chronic (QD1-9) treatment schedule wasfound to be superior for almost all the compounds studied. Significantactivity was obtained for all the diaziridinyl derivatives except thepyrrolidino, No. 43, and piperidino, No. 44, compounds. Some of the datavariation may be attributable to the problem of reproducibly injectingsuspensions of the more insoluble compounds.

                                      TABLE VIII                                  __________________________________________________________________________    INTRAPERITONEAL LYMPHOID LEUKEMIA L1210 ACTIVITY                              Comp.                                                                             QD1-9.sup.(a)    Q4D(1,5,9).sup.(b)                                                                           Day 1.sup.(c)                             No. Exp. No.                                                                           OD.sup.(d)                                                                        T-C.sup.(e)                                                                       ILS.sup.(f,g)                                                                     Exp. No.                                                                           OD T-C                                                                              ILS Exp. No.                                                                           OD T-C                                                                              ILS                            __________________________________________________________________________    26  7968 5.0 -2.9                                                                              69  7691 12.5                                                                             -1.8                                                                             93  8744 5.0                                                                              -0.9                                                                             42                                 7967 5.0 -4.4                                                                              37  7982 12.5                                                                             -2.4                                                                             52  8743 10.0                                                                             -3.3                                                                             39                             31  8769 6.0 -3.5                                                                              29  8698 6.2                                                                              -1.8                                                                             21  8757 12.5                                                                             -3.2                                                                             21                                 8770 1.5 -0.2                                                                              11  8697 3.1                                                                              -2.2                                                                             7   8758 6.2                                                                              -3.3                                                                             4                              33  8857 0.4 -4.5                                                                              115 8546 0.5                                                                              -1.7                                                                             161(3)                                                                            8757 1.0                                                                              -1.7                                                                             95                                 8856 0.3 -0.3                                                                              100 8769 0.02                                                                             -2.8                                                                             124(2)                                                                            8758 0.5                                                                              -0.9                                                                             26                             34  8343 0.2 -1.6                                                                              73(1)                                                                             8343 0.8                                                                              -1.6                                                                             57  8744 0.7                                                                              -3.4                                                                             34                                 8004 0.2 -2.5                                                                              61  7860 0.4                                                                              -1.0                                                                             56  8743 0.7                                                                              -2.8                                                                             19                             35  8004 1.5 -3.3                                                                              112(2)                                                                            7860 3.0                                                                              -2.3                                                                             75  8744 1.5                                                                              -1.9                                                                             68                                 8005 1.5 -3.3                                                                              91(1)                                                                             7861 1.5                                                                              -1.8                                                                             57  8743 1.5                                                                              -2.2                                                                             54                             36  8005 6.2 -2.9                                                                              48  7861 6.2                                                                              -1.1                                                                             28  8743 12.0                                                                             +0.2                                                                             41                                 8004 6.2 -2.4                                                                              43  7860 6.2                                                                              -0.3                                                                             25  8744 1.5                                                                              -0.1                                                                             17                             37  8741 10.0                                                                              -1.5                                                                              36  8663 12.5                                                                             -1.4                                                                             44  8743 10.0                                                                             -0.8                                                                             11                                 8742 5.0 -1.7                                                                              30  8727 8.2                                                                              -2.9                                                                             25  8744 10.0                                                                             -1.4                                                                             5                              38  5178 0.7 -3.2                                                                              205(2)                                                                            7861 0.7                                                                              -2.0                                                                             131 8647 1.5                                                                              -3.8                                                                             175(5)                             8005 0.7 -3.0                                                                              134(3)                                                                            7860 0.7                                                                              -0.3                                                                             70  8743 1.5                                                                              -3.4                                                                             119(3)                         39  8647 4.0 -2.7                                                                              206(5)                                                                            8450 4.0                                                                              -0.3                                                                             90  8743 4.0                                                                              -3.2                                                                             173(4)                             8648 2.0 -2.1                                                                              133(2)                                                                            8454 8.0                                                                              -2.0                                                                             79  8744 2.0                                                                              -2.3                                                                             89                             40  8861 10.0                                                                              -4.9                                                                              92  8663 8.0                                                                              -2.7                                                                             93  8743 10.0                                                                             -1.6                                                                             21                                 8742 10.0                                                                              -2.7                                                                              91  8727 8.0                                                                              -2.8                                                                             32  8744 10.0                                                                             -1.8                                                                             15                             41  8741 0.75                                                                              -1.6                                                                              39  8740 3.0                                                                              - 0.4                                                                            120(1)                                                                            8743 1.5                                                                              +0.4                                                                             21                                 8742 0.37                                                                               0.0                                                                              16  8656 3.0                                                                              -1.0                                                                             73  8744 1.5                                                                              -2.2                                                                             15                             42  8742 1.5 -2.4                                                                              114(1)                                                                            8663 0.4                                                                              -0.9                                                                             57  8744 1.0                                                                              -0.9                                                                             26                                 8826 1.5 -3.6                                                                              111(1)                                                                            8727 0.2                                                                              +4.0                                                                             19  8743 0.2                                                                              -2.6                                                                             -5                             43  8769 2.0 -2.7                                                                              24  8698 6.2                                                                              -2.3                                                                             43  8757 4.0                                                                              -1.0                                                                             25                                 8770 2.0 -2.2                                                                              21                 8758 2.0                                                                              -0.9                                                                             8                              44  8883 10.0                                                                              -2.2                                                                              28  8739 35 -2.4                                                                             26  8757 20.0                                                                             -1.7                                                                             16                                                  8697 12.5                                                                             -3.0                                                                             22  8758 80.0                                                                             -2.7                                                                             8                              45  8741 3.0 -1.8                                                                              59  8727 6.0                                                                              -3.8                                                                             104(2)                                                                            8743 3.0                                                                              -0.7                                                                             21                                 8742 3.0 -3.6                                                                              44  8663 4.0                                                                              -2.5                                                                             84  8744 0.7                                                                              -0.9                                                                             8                              46  8769 6.0 -5.7                                                                              64  8697 6.0                                                                              -2.6                                                                             49  8757 12.5                                                                             -3.0                                                                             42                                 8770 1.5 -2.7                                                                              32  8698 6.0                                                                              -2.6                                                                             44  8758 3.1                                                                              -1.6                                                                             -14                            47  8539 1.0 -1.7                                                                              147 8320 4.0                                                                              -2.1                                                                             180(3)                                                                            8743 4.0                                                                              -0.7                                                                             52                                 8647 0.5 -1.7                                                                              114(1)                                                                            8739 2.0                                                                              -1.5                                                                             165(4)                                                                            8744 4.0                                                                              -3.4                                                                             21                             __________________________________________________________________________     .sup.(a) Day 1-9 treatment schedule (9 injections)                            .sup.(b) Day 1,5,9 treatment schedule (3 injections)                          .sup.(c) Day 1 only treatment schedule (1 injection)                          .sup.(d) Optimum dose (mg/kg/injection)                                       .sup.(e) Average weight change of test group minus average weight change      of control group in grams on day 5.                                           .sup.(f) Percentage increase in life span of treated animals [(treated        survival + control survival) × 100%] -100%                              .sup.(g) Number of animals alive per 6 test animals on the day of             termination of the experiment (L1210 and P388day 30; B16 melanoma and         ependymoblastomaday 60)                                                  

                                      TABLE IX                                    __________________________________________________________________________    INTRAPERITONEAL LYMPHOCYTIC LEUKEMIA P388 AND B16                             MELANOCARCINOMA ACTIVITY.sup.(a)                                              Compound                                                                            Lymphocytic Leukemia P388                                                                      B16 Melanocarcinoma                                    No.   Exp. No.                                                                           OD.sup.(b)                                                                        T-C ILS Exp. No.                                                                           OD T-C ILS                                        __________________________________________________________________________    26                     301  5.0                                                                              -1.0                                                                              70                                                                300  5.0                                                                              -0.7                                                                              48                                         31    5282 3.0 -2.3                                                                              57  302  0.7                                                                              -0.3                                                                              18                                               5281 6.0 -2.5                                                                              54  303  0.7                                                                               0.0                                                                               9                                         33    5281 0.2 -1.7                                                                              171(1)                                                                            302  0.1                                                                              -0.5                                                                              31                                               5439 0.2 -1.9                                                                              156 303  0.02                                                                             -0.1                                                                              19                                         34    5112 0.2 -3.6                                                                              95  301  0.4                                                                              -0.7                                                                              39                                               5111 0.1 -3.3                                                                              69  294  0.05                                                                             -3.1                                                                              29                                         35    4902 1.5 -2.6                                                                              160 300  0.4                                                                              -0.3                                                                              26                                               4903 0.7 -2.0                                                                              103 301  0.7                                                                              -0.4                                                                              24                                         36    5111 6.0 -3.0                                                                              151(2)                                                                            288  5.0                                                                              -0.5                                                                              36                                               5112 6.0 -3.3                                                                              108 287  5.0                                                                              -2.9                                                                              32                                         37    5281 5.0 -0.4                                                                              64  302  10.0                                                                             -1.0                                                                              36                                               5282 10.0                                                                              -2.0                                                                              49  303  5.0                                                                              -0.1                                                                              11                                         38    4903 0.4 -2.6                                                                              175(3)                                                                            288  0.4                                                                              -0.6                                                                              61                                               4902 0.4 -2.3                                                                              160(2)                                                                            287  0.4                                                                              -2.7                                                                              51                                         39    5262 4.0 -3.4                                                                              143(6)                                                                            301  4.0                                                                              -2.5                                                                              50                                               5405 4.0 -3.5                                                                              141(4)                                                                            314  6.0                                                                              -3.0                                                                              42                                         40    5281 2.0 -2.3                                                                              85  302  8.0                                                                              -1.4                                                                              37                                               5282 2.0 -1.1                                                                              71  303  0.5                                                                              +0.5                                                                              22                                         41    5282 1.0 -2.7                                                                              74  303  0.2                                                                              +0.3                                                                              16                                               5281 0.5 -1.8                                                                              37  302  0.2                                                                              -0.5                                                                              12                                         42    5439 1.6 -2.4                                                                              162 302  0.8                                                                              -2.3                                                                              47                                               5440 0.8 -2.7                                                                              145 303  0.8                                                                              -1.2                                                                              31                                         43    5282 1.0 -1.8                                                                              72  303  2.0                                                                              +0.8                                                                              28                                               5281 0.2 -4.3                                                                              55  302  2.0                                                                              -0.5                                                                              18                                         44    5525 5.0 +2.3                                                                              46  317  5.0                                                                              -1.5                                                                              21                                               5526 10.0                                                                              -1.5                                                                              77  316  2.5                                                                              -2.3                                                                              -3                                         45    5282 1.0 -2.5                                                                              95  302  1.0                                                                              -1.1                                                                              40                                               5281 1.0 -0.5                                                                              78  303  1.0                                                                              -0.1                                                                              37                                         46    5525 0.5 -0.1                                                                              52  302  1.5                                                                              -1.8                                                                              24                                               5281 0.7 -0.4                                                                              48  303  0.7                                                                              +1.0                                                                              17                                         47    5263 0.5 -0.8                                                                              149(3)                                                                            301  0.5                                                                              -0.6                                                                              22                                               5262 1.0 -2.4                                                                              143(6)                                                                            300  0.2                                                                              +0.1                                                                              22                                         __________________________________________________________________________     .sup.(a) QD1-9 treatment schedule                                             .sup.(b) See footnotes in Table VIII for definitions                     

                                      TABLE X                                     __________________________________________________________________________    INTRACEREBRAL ANTITUMOR ACTIVITY                                              Compound                                                                            Ependymoblastoma.sup.(a)                                                                       Lymphoid Leukemia L1210.sup.(b)                        No.   Exp. No.                                                                           OD.sup.(c)                                                                        T-C ILS Exp. No.                                                                           OD T-C ILS                                        __________________________________________________________________________    26    152  10.0                                                                              -2.5                                                                              294(1)                                                                            35   5.0                                                                              -2.0                                                                              39                                               153  5.0 -1.9                                                                              143(1)                                                                            36   10.0                                                                             -1.3                                                                              25                                         31    169  0.8 +0.1                                                                              49                                                               168  1.5 -1.3                                                                              40                                                         33    177  0.2 -2.7                                                                              215(4)                                                                            43   0.3                                                                               0.0                                                                              60                                               155  0.2 -2.0                                                                              177(6)                                                                            42   0.3                                                                              -1.0                                                                              59                                         34    174  0.2 -1.5                                                                              219(5)                                                                            38   0.4                                                                              -0.3                                                                              68                                               175  0.4 -1.8                                                                              218(4)                                                                            33   0.2                                                                              +0.3                                                                              60                                         35    175  1.5 -2.0                                                                              218(2)                                                                            30   3.0                                                                              -2.2                                                                              44                                               174  1.5 -2.3                                                                              187(2)                                                                            31   3.0                                                                              -1.8                                                                              40                                         36    152  10.0                                                                              -1.9                                                                              259(2)                                                           153  5.0 -1.2                                                                              143(1)                                                     37    166  6.0 -2.3                                                                              93                                                               165  3.0 -0.3                                                                              46                                                         38    152  1.5 -3.0                                                                              83  43   0.75                                                                             +0.4                                                                              57                                               174  1.5 -1.0                                                                              70  30   0.75                                                                             -1.8                                                                              48                                         39    155  1.0 -0.4                                                                              99(1)                                                                             39   4.0                                                                              -1.6                                                                              43                                               156  4.0 -3.3                                                                              53  40   4.0                                                                              -1.0                                                                              41                                         40    166  2.0 -3.2                                                                              64  42   2.5                                                                              -1.5                                                                              16                                               165  1.0 -1.4                                                                              50  43   10.0                                                                             -1.8                                                                              16                                         41    177  1.0 -2.3                                                                              215(5)                                                                            43   3.0                                                                              -0.4                                                                              72.sup.(d)                                       176  0.5 +0.4                                                                              190(4)                                                                            42   3.0                                                                              +0.1                                                                              56.sup.(d)                                 42    166  0.4 -1.6                                                                              84  43   4.0                                                                              -0.7                                                                              45                                               165  0.2 -1.2                                                                              69  42   2.0                                                                               0.9                                                                              32                                         43    171  1.6 -1.9                                                                              101                                                              170  3.1 -1.4                                                                              47                                                         44    171  6.2 -2.4                                                                              68                                                               170  12.5                                                                              -3.1                                                                              46                                                         45    165  0.5 -1.2                                                                              64  43   8.0                                                                              -0.3                                                                              30.sup.(d)                                       166  2.0 -4.6                                                                              48  42   6.0                                                                              -1.0                                                                              14.sup.(d)                                 46    168  1.5 -1.8                                                                              134                                                              169  0.4 +0.8                                                                              82                                                         47    155  1.0 -1.2                                                                              75  39   2.0                                                                              -1.4                                                                              43                                               174  1.0 -3.0                                                                              75  41   1.5                                                                              -1.4                                                                              41                                         __________________________________________________________________________     .sup.(a) QD1-5 treatment schedule                                             .sup.(b) QD1-9 treatment schedule unless otherwise indicated                  .sup.(c) See footnotes Table VIII for definitions                             .sup.(d) Q4D(1,5,9) treatment schedule                                   

                  TABLE XI                                                        ______________________________________                                        AZIRIDINYLBENZOQUINONES WITH OUTSTANDING                                      ACTIVITY IN TWO OR MORE TUMOR SYSTEMS                                         Compound                                                                              ILS (%)                                                               No.     IP L1210  P388    B16   IC L1210                                                                             EM                                     ______________________________________                                        26                        70           294(1)                                  9      169       138(1)  70    84     277(5)                                 33      161(3)    171(1)        60     177(6)                                 34                              68     219(5)                                  0      112(2)    160                  218(2)                                 36                151(2)               259(2)                                 38      205(2)    175(3)  61    57                                            39      206(5)    143(6)  50            99(1)*                                41      120(1)                  72     215(5)                                 42      114(1)    162                                                         47      180(3)    143(3)                                                      ______________________________________                                         *Repeat tests gave ILS values of 186(6) and 53%                          

Superior activity was obtained with several derivatives among which thedihydroxypropylamino and hydroxyethylamino compounds were outstandingwith maximum ILS values in excess of 200% being observed. Thetheoretical maximum ILS values in a fully acceptable experiment rangesbetween 173 and 275% consistent with the control animal death limits setat 8 to 11 days and experiment termination on day 30. Multiple cures,defined as survivors on day 30 in the L1210 system, were observed forboth compounds. Excellent activity was also observed with compounds Nos.38 and 39 on the day 1 and Q4D treatment schedules. The day 1 schedulewas generally the poorest of the three studied. Several compounds showedmaximum activity with intermittent (Q4D) treatment.

Excellent activity was observed in the leukemia P388 system on thechronic treatment schedule (Table IX). Again, compounds 38, 39 and 47were very active producing multiple cures (30 day survivors). As mightbe expected, compounds with outstanding L1210 activity usually possessedexcellent P388 activity (Table XI). All the compounds studied in thisseries (Table IX) were active against leukemia P388, including themonoaziridinyl derivative, No. 31.

Only three compounds in the series were reproducibly active in the B16melanoma tumor model (Table IX). Once again Nos. 38 and 39 proved activeas did the 2'-methylaziridinyl analog, No. 26. The aziridinyl analog ofNo. 26 (No. 34) approached minimal reproducible activity as did No. 42,the N-methyl derivative of No. 38, and the 3'-hydroxypiperidino analog,No. 45.

Table X shows the data obtained in the intracerebrally implanted tumorsystems. Only those compounds which had given L1210 intraperitoneal ILSvalues greater than 50% were tested in the intracerebral L1210 system.While nine of eleven compounds tests were reproducibly active, theparent amino compound (No. 33), and mono- (no. 34) and dimethylamino(No. 41) analogs were the most active against IC L1210 leukemia.Compounds Nos. 38, 39 and 47 which produced cures in the IP L1210 modelgave activity levels which may be consistent with the inhibition ofsystemic disease produced by migration of L1210 cells from the implantsite. The marginal IC L1210 activity associated with these threecompounds, which produce long term survivors in the IP system, may berelated to their hydrophilic nature, considering the log P values inTable VII, and a reduced ability to cross the blood-brain barrier.

All of the compounds in this study were reproducibly active against theependymoblastoma or EM murine brain tumor system. It is probably moreuseful to compare cures, in the terms of 60 day survivors, rather thanILS values in the EM model, since the ILS for a highly active compoundis very dependent on the life span of the control animals which normallyranges from 17-21 days. The best IC L1210 active derivatives (Nos. 33,34 and 41) were again among the most active materials against the EMtumor system. The ethyl- (No. 34) and propylamino (No. 36) compounds andthe 2'-methylaziridinyl analog of No. 34 (compound 26) were also highlyactive. The hydrophilic dihydroxypropylamino compound (No. 39), whichwas curative in the IP L1210 and P388 leukemia systems, was active butgave a large spread in ILS values (53-186%). For this reason, No. 39should be considered to have moderate to good EM activity. Again, itshydrophilic character may present transport problems in this IC tumorsystem. It is noteworthy that the majority of the most active compoundsin the EM system are the simple monoalkylamino derivatives and theirparent amine (No. 33).

Thus it is seen that several general points can be made about thediaminodiaziridinylbenzoquinone family. Its members have good antitumoractivity, usually in several tumor systems. They are very potent withmost optimum doses in the 0.1 to 5.0 mg/kg range on a chronicadministration schedule. Therapeutic ratios (highest active dose dividedby lowest active dose) were somewhat dependent upon the tumor system.For the most active compounds, therapeutic ratios averaged 4, 8 and 2 inIP L1210, P388 and B16 melanoma, respectively. These values wereapproximately 2 and 8 in the IC L1210 and ependymoblastoma tumorsystems. These compounds are alkylating agents giving a positive4-(p-nitrobenzyl)pyridine (NBP) test.

Several qualitative structure-activity relationships are apparent. Thecompounds that are most active in the IP ascites tumor systems are themost polar, water soluble derivatives (Nos. 38, 39, 47). The parentamino compound No. 33, however, is also active and is so insoluble inany solvent that an ultraviolet spectrum was non-attainable. Among themonoalkylamines, activity appears to peak with the ethyl derivative (No.35) in the L1210 series. A significant reduction in activity is notedfor the propyl (No. 36) and butyl (No. 37) analogs. Leukemia P388 isusually a more sensitive system than L1210. In this series, however,L1210 activities were often equal to or greater than P388 values withthe same compounds active against both tumors. Multiple cures wereobtained in both systems.

Although several derivatives possess substantial reproducible B16melanoma activity, this tumor is the most refractory of those studied inthis investigation. The relationship between outstanding ascites tumoractivity and B16 activity for Nos. 38 and 39 (Table XI) does not holdfor several other compounds (Nos. 26, 47).

In the intracerebral systems, the trend toward activity with the morelipophilic compounds is apparent. The propyl derivative (No. 36) isstill very active in the EM system and even the butyl analog hasactivity. These two groups greatly reduced L1210 activity relative totheir lower carbon number congeners. Exceptionally high IP L1210activity does not guarantee good activity in the IC L1210 system (Nos.35, 42, 47). The significant activity of the parent compound (No. 33) aswell as its methyl (No. 34) and dimethyl (No. 41) derivatives in thenormally refractory IC L1210 system is noteworthy. The polar, watersoluble compounds were generally less active in the EM system than thenonhydroxylated derivatives.

A comparison of activity in all tumor systems (Table XI) shows thatthose compounds which are best in the IP systems are not the most activein the IC systems. The dihydroxypropylamino analog (No. 39, which givesmultiple long term survivors against the leukemia L1210 and P388 tumorsis probably the most active compound in the IP ascites systems. Thehydroxyethylamino Nos. 38 and 47 are also superior in these systems. Inthe EM system, the unsubstituted amino compound No. 33, and severalalkylamino analogs (Nos. 34, 35, 36 and 41) are most active. Theethylcarbamate derivative, No. 9, is included in Table XI forcomparison. While this compound does not give multiple cures in theascites systems, it is curative in the EM system and has verysubstantial activity in all the other tumor systems studied. The highactivity of the insoluble amino derivative No. 33 suggests thepossibility that No. 33 might be the active agent and that the urethane9 and the various active acylamino derivatives, such as No. 27, serve assolubility enhancing groups which facilitate drug transport to the tumorcell and then produce No. 33 by hydrolysis. There is, however, noevidence for this at the present time.

Since retention of CNS antitumor activity with an improvement in watersolubility by the use of non-ionic hydrophilic groups was a majorobjective of this investigation, a comment regarding the watersolubilities and partition coefficients of the diaziridinylbenzoquinones(Table VII) is in order. The unsubstituted, parent amino derivative (No.33) is an exceptionally insoluble material, not only in water, but inall solvents studied. The monoalkylamino compounds also have verylimited water solubility. The addition of a second alkyl group (e.g.,No. 41 vs No. 34) which might be expected to lower water solubility,actually increases the solubility greatly. This effect is also apparentin a comparison of No. 42 and No. 38. In both of these cases a dramaticdecrease in melting point takes place upon addition of the second alkyl,in this case methyl, group. The increase in water solubility, therefore,may be due to a decrease in hydrogen bonding properties and a resultingdecrease in the crystal forces in the solid state.

The partition coefficients of a few of these compounds were determined(Table VII). Initial calculated approximations of the partitioncoefficients in this series were made using the log P value ofp-benzoquinone and substitutent pi values in order to help determinewhich molecules to synthesize. The values measured after synthesis werequalitatively in the right order but contained large quantitativedifferences. This was expected because attempts to calculate knownquinone partition coefficients values from a parent quinone plus pivalues produced very poor results.

The partition coefficient of No. 9 was measured and found to have a logP value of 0.05. The water solubility of No. 9 is about 0.5 mg/ml. Acomparison of log P values and water solubilities (Table VII) shows thata general qualitative correspondence between hydrophilicity and watersolubility exists. However, no quantitative relationship between the twois apparent for the five derivatives with log P values.

In the following examples, all melting points are uncorrected and wererecorded on a Thomas-Hoover capillary melting point apparatus. Elementalanalyses were performed by NIAMDD, NIH, Bethesda, Md. Fluoranil,ethyleneimine and 2-methylethyleneimine were obtained commercially. Whenseveral compounds were prepared by comparable procedures, only onerepresentative example is included. New compounds were identified by nmrand ir spectroscopy. In the tetraamino derivatives, the methyleneprotons of the aziridine rings usually occurred as a singlet at δ2.0-2.2in CDCl₃ or DMSO-d₆. This absorption appeared at δ2.40 (CDCl₃) forcompound 11. Carbonyl absorption frequencies (nujol mull) occurred inthe range 1620-1640 cm⁻¹ for the tetraamino compounds. Satisfactoryelemental analyses (+0.4% of calculated values) are indicated byelemental symbols in Table VII. Room temperature water solubilities weredetermined by the incremental addition (pipet) of water to a weighedamount of compound with shaking. The solubility values in Table VII mustbe considered as approximate.

2,5-DIAZIRIDINYL-3,6-DIFLUORO-1,4-BENZOQUINONE (32, (GENERAL PROCEDUREFOR 25)

A solution of ethyleneimine (33.3 g, 0,77 mol) in dry tetrahydrofuran(100 ml) was added dropwise to a stirred solution of fluoranil (25.0 g,0.14 mol) in tetrahydrofuran (100 ml) cooled by an ice water bath. Thecold solution was stirred for 40 min, cold water (100 ml) was added. Theprecipitate was filtered, washed thoroughly with water and dried.Recrystallization from toluene gave 19.4 g (62%) of red crystals, mp220° (lit. mp 230°, 212°)

2,5-DIZAIRIDINYL-3,6-DIAMINO-1,4-BENZOQUINONE (33)

A solution of compound 32 (0.56 g, 2.5 mmol) in tetrahydrofuran (100 ml)was saturated with dry ammonia gas at 2°. This solution was transferredto a pressure bottle and was heated overnight at 50°-55°. After coolingto room temperature, the bottle was opened carefully. The precipitatewas filtered, washed with water, and dried to give 0.40 g (73%) of greencrystals, mp 220°.

2,5-DIAZIRIDINYL-3,6-BIS(ETHYLAMINO)-1,4-BENZOQUINONE (35) (GENERALPROCEDURE FOR 26, 34, 36-40, 42, 44, 45)

Anhydrous ethylamine (25 ml, 17.3 g, 380 mmol) was added to a stirredice cold solution of compound 32 (2.0 g, 9 mmol) in 600 ml oftetrahydrofuran (methylamine used in the preparation of compound 34 wasa 40% aqueous solution). The reaction mixture was stirred at 2° for 2 hrand then at 23° for 80 hr. Evaporation of the solvent in vacuo gave adark green solid which was washed with ice water and dried in vacuo overKOH pellets. Recrystallization from ethanol gave 1.40 g (57%) of darkgreen prisms, mp 156°-157°.

2,5-DIAZIRIDINYL-3,6-BIS(DIMETHYLAMINO)-1,4-BENZOQUINONE (41)

Dimethylamine hydrochloride (16.3 g, 0.2 mol) was slowly added to astirred ice cold methanolic sodium methoxide solution (4.6 g, 0.2 mol Nain 100 ml methanol). The resulting mixture was stirred at roomtemperature for 0.5 hr and a solution of compound 32 (1.13 g, 0.005 mol)in tetrahydrofuran (300 ml) was added. The reaction mixture was stirredovernight at ambient temperature. The solvent was evaporated in vacuo togive a dark solid which was extracted with benzene. The solid obtainedfrom evaporation of the benzene was recrystallized from hexane to give1.1 g (59%) of green solid, mp 110°-112°.

2,5-DIAZIRIDINYL-3,6-DIPYRROLIDINO-1,4-BENZOQUINONE (43)

Compound No. 32 (1.13 g, 5 mmol) and pyrrolidine (2.0 ml, 1.7 g, 24mmole) were added to benzene (250 ml) and the mixture was stirred at 23°for 3 days. The dark solution was heated to 50°-60° for 2 hr. Theresulting precipitate was filtered and discarded. The filtrate wasconcentrated to give a brown solid which was recrystallized fromtetrahydrofuran/hexane to give 1.05 g (64%) of brown product, mp 160°.

2,5-DIAZIRIDINYL-3,6-DIMORPHOLINO-1,4-BENZOQUINONE (46) AND2,5-DIAZIRIDINYL-3-FLUORO-5-MORPHOLINO-1,4-BENZOQUINONE (31)

Morpholine (1.75 g, 20 mmol) was added to an ambient solution ofcompound 32 (1.13 g, 5 mmol) in tetrahydrofuran (250 ml). After stirringovernight, the solvent was removed in vacuo. The solid was washed withwater, and recrystallized from benzene to give 0.50 g of compound 46 asa brown solid, (28%) mp 224°. Addition of the recrystallization filtrateto hexane gave 0.75 g (51%) of gray compound 31, mp 157°.

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the method describedwithout departing from the spirit and scope of the invention orsacrificing its material advantages, the forms hereinbefore describedbeing merely the preferred embodiments thereof.

It is claimed:
 1. A chemotherapeutic method for the treatment ofmalignant tumors located in the central nervous system of a humanpatient which comprises administering to said patient anantitumor-effective amount of(a)2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone.
 2. Themethod of claim 1 wherein the administration of the compound isparenteral.
 3. The method of claim 2 wherein the administration is byintravenous injection.
 4. The method of claim 1 wherein the tumor is abrain tumor.
 5. The method of claim 4 wherein the tumor is aGlioblastoma or an Anaplastic Astrocytoma.
 6. The method of claim 1wherein the tumor is a Melanoma or a Leukemia.
 7. The method of claim 1wherein the compound is administered in combination with other antitumoreffective compounds.
 8. The method of claim 1 wherein the tumor is aprimary tumor.